Fig 1.
Mini-gene analysis of c.1679+1634A>G mutation.
a) Schematic of pMG-in12-WT plasmid comprising a contiguous ~1.5kb region of CFTR containing part of intron 12, exon 13 and part of intron 13 cloned between the 5’ and 3’ exons of the pSPL3 vector. Exons are shown as boxes, introns as lines, and predicted transcript as thicker line shown underneath. Arrows below transcript represent pspRTfw and pspRTrv primers for RT-PCR. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in12-WT and size markers (unfilled peaks). c) Schematic of pMG-in12-A>G mutation which differs from pMG-in12-WT by the A>G change shown with dotted arrow. The A>G change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in12 vector. Arrows above DNA represent pspSEQfw and in12Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in12-A>G. e) Schematic of pMG-in12-A>G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in12-A>G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in12-A>G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in12-A>G and pTandem-in12. The top band is generated from untargeted DNA, the bottom band is generated from DNA containing the targeted deletion, and the middle band is most likely heteroduplexes formed during the late stages of PCR; similar bands have been reported during PCR analysis of the CF-causing c.1521_1523delCTT deletion and verified as heteroduplexes [32]. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in12-A>G and pTandem-in12 vector.
Fig 2.
Mini-gene analysis of c.3140-26A>G mutation.
a) Schematic of pMG-in19-WT plasmid comprising a contiguous ~2.0 kb region of CFTR containing part of intron 18, exon 19, all of intron 19, exon 20 and part of intron 20 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-WT. c) Schematic of pMG-in19-A>G mutation which differs from pMG-in19-WT by the A>G change shown with dotted arrow. The A>G change extends exon 20 by 25 bp which shifts the reading frame and eventually results in-frame stop codon (TGA). The triangles indicate target sites of Cas9/gRNAs encoded by pTandem-in19 vector. Arrows above DNA represent in19Ufw and pspSEQrv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-A>G. e) Schematic of pMG-in19-A>G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in19-A>G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A>G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A>G and pTandem-in12. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in19-A>G and pTandem-in19 vector.
Fig 3.
Mini-gene analysis of c.3718-2477C>T mutation.
a) Schematic of pMG-in22-WT plasmid comprising a contiguous ~3.2 kb region of CFTR containing part of intron 22, exon 23, and part of intron 23 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in22-WT. c) Schematic of pMG-in22-C>T mutation which differs from pMG-in22-WT by the C>T change shown with dotted arrow. The C>T change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in22 vector. Arrows above DNA represent pspSEQfw and in22Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in22-C>T. e) Schematic of pMG-in22-C>T mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in22-C>T measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C>T only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C>T and pTandem-in22. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in22-C>T and pTandem-in22 vector.